Ortiz-Leduc 9415446 Molecular chaperones are involved in the translocation, folding, and assembly of polypeptides in chloroplasts and mitochondria. This proposal is based on the premise that a novel 60,000 Mr polypeptide that accumulates in chloroplasts of temperature- stressed Euglena gracilis is a chaperone of the conserved cpn60 family. In this regard, the 60,000 Mr species appears to be a functional chaperone at the normal growth temperature (23 C) but nonfunctional at elevated temperatures. Thus, a failed chaperone response could be at the rootcause for the irreversible loss of chlorophyll (and photosynthesis) observed at the moderately elevated temperature of 33 C--a phenomenon known as heat bleaching. Western blot analysis will be used to confirm the identity of the novel 60,000 Mr species in bleaching Euglena using commercial antibodies to bacterial and mitochondrial cpn60. We will carry in vivo and in organello pulse-labeling experiments to study the ability of chloroplast cpn60 to associate with newly synthesized polypeptides at permissive and nonpermissive temperatures. We will also isolate cpn60 from Euglena chloroplast extracts using size- exclusion chromatography and density-gradient centrifiguration. Finally, we will attempt to show that isolated Euglena cpn60 can mediate the in vitro reactivation of chemically denatured dihydrofolate reductase at normal and heat-shock temperatures. If, as expected, cpn60 fails to perform as a chaperone at high temperatures, future works will focus on the molecular basis of Euglena cpn60 dysfunction using recombinant DNA technology. %%% The proposed research focuses on a group of proteins--the chaperones--that play a vital and fundamental role in the cells of living organisms from bacteria to plants to humans. Chaperones normally assist other proteins during folding, during transport across biological membranes, and during assembly of large multiprotein structures. In addition, chaperones appear to protec t cellular structures against damage from heat stress when organisms experience potentially lethal temperatures. The process of photosynthesis in plants is notoriously sensitive to high temperatures. The unicellular alga Euglena gracilis, in particular, appears to be unusually sensitive to temperature because the algo loses chlorophyll and chloroplast function when cultures are grown at the moderately elevated temperature of 33 C. Unfortunately, research on plant chaperones and their role during periods of heat-stress has received proportionately less attention than chaperones in bacterial, yeast, and mammalian systems. Advances in agriculture are intimately tied to the ability of plants to harvest light energy and convert it to biomass. In this regard, cereals, an important crop in Plain States, are also sensitive to heat stress and fail to develop functional chloroplasts when seedlings are grown at 33 C. Using Euglena as a simple model system, we want to investigate why chloroplasts are sensitive to high temperatures. The failure of chloroplast chaperones to function properly at 33C could explain why photosynthetic structures are not adequately protected from heat- stress.
